Summary Filoviruses, which include the Ebola viruses (EBOV) and Marburg viruses (MARV) are non-segmented, negative-sense RNA viruses (NNSVs) that cause severe disease. Fundamental to filovirus replication is the viral RNA synthesis machinery. Viral proteins, in concert with cis- and trans-acting factors, regulate the viral RNA-dependent RNA polymerase (RDRP). However, we lack a comprehensive understanding as to how such factors works together to regulate viral RNA synthesis. This project (Project 1) will build upon several novel observations by the Basler lab and upon data generated in Projects 2 and 3 of this program to determine the mechanisms by which specific cis-acting sequences and trans-acting proteins modulate filovirus replication. It will also define the functional consequences of these mechanisms for replication. One observation was the identification of short upstream open reading frames (uORFs) in the 5' UTRs of the EBOV VP35, VP30, VP24 and L mRNAs as cis-acting regulators of protein expression. The L uORF was further demonstrated to modulate L protein translation in response to cell stress and eIF-2? phosphorylation levels. We hypothesize that this provides a mechanism by which the virus regulates expression of its RDRP machinery in response to the innate immune status of the host cell. We will use reporter gene-based approaches and ribosomal footprinting methods to determine how filoviral UTRs and uORFs modulate mRNA translation and design mutant recombinant EBOVs to assess these functions in the context of infection. A second finding was the identification of several previously unrecognized sites in both EBOV and MARV where substantial mRNA editing occurs. This is similar to the previously recognized editing of the EBOV glycoprotein (GP) mRNA, where the viral RDRP adds non-template encoded ?As? at a site that corresponds to a specific polyU stretch on the template genomic RNA. Because the determinants of editing frequency by the filoviral RDRP remain incompletely defined and because the biological significance of the edited mRNAs' translation products is unclear, we will use a reconstituted viral RDRP complex and deep sequencing approaches to define cis-acting elements that regulate editing events. Finally, we and others have identified trans-acting factors such as the cellular proteins LC8, PACT and, DRBP76 as modulators of EBOV RNA synthesis. These observations support the hypothesis that host factors will modulate viral RDRP function in significant ways. Studies performed in Project 2 of this application will define interactions among the viral proteins that comprise the EBOV RDRP complex. Project 3 will comprehensively evaluate host factors for modulation of virus replication. Project 1 will use established transfection-based assays of viral RNA synthesis to evaluate the impact of LC8, PACT and, DRBP76 and newly identified interactions on the viral RDRP and define mechanisms of action. In total, these studies will provide substantial new insights into mechanisms regulating filovirus RNA synthesis that may be exploited for therapeutic development.